Anaerobic biological degradation of hydrocarbons

ABSTRACT

The invention relates to a method for the anaerobic biological degradation of aromatic hydrocarbons (in particular benzene), and to a specific mixture and the use thereof for this degradation. According to the invention, the anaerobic biological degradation of aromatic hydrocarbons present at a contaminated location is stimulated and stabilized by the use of a combination of humic acids and/or anthraquinone-2,6-disulfate and nitrate, which is added to anaerobic bacterial populations.

The invention relates to a method for the anaerobic biologicaldegradation of hydrocarbons, specifically aromatic and aliphatichydrocarbons, and to a specific mixture and the use thereof for thisdegradation.

In soil remediations, for the purpose of degradation of aromatichydrocarbons, such as benzene, typically use is made of aerobicdegradation. The net reaction equation for this degradation can berepresented (for benzene) as follows:2C₆H₆+15O₂ →12CO₂+6H₂O  (1)

Compressed air injection is the most commonly used method to effect suchdegradation. In addition, methods are known where oxygen releasecompounds (ORC) are introduced into the soil. Examples of suchcomponents are hydrogen peroxide, ozone and solids such as magnesiumperoxide (MgO₂). The methods in which oxygen-bearing components areintroduced into the soil are deployed in particular on a smaller scalebut have as a drawback that the components mentioned are chemicallyunstable and/or have a minor bioavailability.

Especially in deep soil systems, certainly if these have a complexstructure, the introduction of oxygen is costly, inefficient anddifficult to carry out.

U.S. Pat. No. 6,432,693 discloses a method for the anaerobic degradationof halogenated organic contaminants and the oxidized forms of organiccontaminants. To that end, a specific solids mixture of metals is used.Because the solids mixture from U.S. Pat. No. 6,432,693 apparently hasdifficulty remaining in solution, it is proposed in that patentpublication to use a chelating agent. As one of the chelating agents,U.S. Pat. No. 6,432,693 suggests humic acid, which is capable of bindingto the dosed metals and can bring them into solution.

Although anaerobic degradation of benzene has been demonstrated insoils, it has been found that this degradation capacity is not presentin many locations. At locations where the anaerobic degradation doesoccur, the process proceeds, for instance, according to the followingnet reaction equations, wherein nitrate, iron, and sulfate,respectively, act as electron acceptor:C₆H₆+6HNO₃ →6CO₂+3N₂+6H₂O  (2)C₆H₆+30Fe(OH)₃→6CO₂+30FeO+30H₂O  (3)4C₆H₆+15H₂SO₄→24CO₂+15H₂S+12H₂O  (4)

However, the reaction rates of anaerobic degradation (2-4) are orders ofmagnitude lower than those of aerobic degradation (1). There is onlylittle known about the mechanisms of anaerobic degradation of benzeneand the bacteria involved in this process. As a consequence, techniquesthat reproducibly result in a fast and stable anaerobic benzenedegradation are lacking, which constitutes a considerable limitation tothe development of biological soil remediation of sites contaminatedwith benzene and other aromatic hydrocarbons. Accordingly, there is aneed for alternative methods for the degradation of benzene and otheraromatic hydrocarbons.

During experiments in a laboratory bioreactor in which an anaerobicculture medium was passed through continuously, it was surprisinglyfound that through the use of a specific mixture of at least oneelectron acceptor and one or more humic acids, this need can be met.Accordingly, the present invention relates to a method for the anaerobicbiological degradation of aromatic hydrocarbons, wherein a combinationof humic acids and nitrate is added to an anaerobic bacterialpopulation.

The anaerobic bacterial populations, which take care of the degradationof the aromatic hydrocarbons, occur naturally in the soil and ingroundwater. What is achieved by dosing the mixture of nitrate and humicacids according to the invention is that the degradation of benzene andother aromatics is stimulated and stabilized under nitrate-reducinganaerobic conditions. A major advantage of the instant finding is thatfor the biodegradation of hydrocarbons in deep anaerobic soil systems,even if they have a complex structure, the costly, inefficient, andcumbersome introduction of oxygen is not necessary anymore.

A very suitable electron acceptor is nitrate, because it is watersoluble, and hence properly doseable in practice, without precipitatesbeing formed. Moreover, nitrate is a very strong electron acceptor. Notonly nitrate, but also other nitrogenous compounds are eligible, inparticular intermediates from the reduction of nitrate, such as nitriteand dinitrogen monoxide (N₂O). In this connection, it is noted that inreaction (2) above, it is not necessarily nitrogen that is formed. It isalso possible that nitrite, N₂O or ammonium (NH₄+) is formed. Nitriteand N₂O in turn can function as electron acceptor.

Further, metal ions, such as Fe(III) and Mn(IV), can be used as electronacceptor. However, the drawback involved is that they form precipitatesand hence are difficult to dose. Moreover, these metals remain presentin the soil. For this reason, the electron acceptor is preferably notbased on iron nor on manganese. In particular, the electron acceptorpreferably does not comprise metallic iron, metallic manganese and/ormanganese salts. More preferably, the electron acceptor is anon-metallic electron acceptor.

Also sulfate can be used as electron acceptor, but sulfate is reduced tosulfide (see reaction equation (4)). Sulfide is toxic and easily formsprecipitates, so that the soil may clog up. Moreover, the oxidizingpower of sulfate is low, which renders it a less strong electronacceptor than nitrate. Therefore, sulfate is less suitable.

Chlorine-containing compounds, specifically chlorate, can also be usedas electron acceptor. Although in principle chlorate has theabove-mentioned advantages of nitrate, chlorate is reduced in the soilto chlorite (ClO₂—), which is not desirable in view of its toxicity.

Surprisingly, it has also been found possible to use certain chlorinatedhydrocarbons as electron acceptor. This can be advantageous specificallyif soil is to be treated which, in addition to being contaminated witharomatics (in particular benzene), is also contaminated with thesechlorinated hydrocarbons. This “combination contamination” often occursin practice. These chlorinated hydrocarbons are preferablyperchlororethylene, trichloroethylene, 1,2-dichloroethane, chlorophenol,chlorobenzoic acid and/or chlorobenzene. In this embodiment, it issufficient to introduce the humic acids into the soil, since theelectron acceptor is already present in it. If desired, also anadditional amount of the above-mentioned electron acceptors, inparticular nitrate, can be supplied.

Without wishing to be bound to any theory, it is supposed that thedegradation of the aromatics according to the invention proceedsaccording to either of the following two hypothetical routes.

According to the first hypothetical route, it is possible that humicacids function as a so-called electron shuttle between bacterium 1 andbacterium 2 in the diagram below and where (for instance) nitratefunctions as terminal electron acceptor: Bacterium 1: aromatic → CO₂ +H₂O + e⁻ oxidized humic acid + e⁻ → reduced humic acid (*) Bacterium 2:reduced humic acid (*) → oxidized humic acid + e⁻ NO₃ ⁻ + e⁻ → N₂

The asterisk (*) here indicates that the product of bacterium 1 is usedby bacterium 2. Although it is plausible that more than one type ofbacterium is involved in the degradation, the possibility that allprocesses are carried out in one type of bacterium cannot be ruled out.According to an alternative hypothetical degradation mechanism, humicacids are used as an electron donor and (for instance) nitrate aselectron acceptor: Bacterium Aromatic + → CO₂ + H₂O + reduced humic acidoxidized humic acid + e⁻ NO₃ ⁻ + e⁻ → N₂

In this case, the humic acids provide for the induction of enzymes thatare involved in the degradation of benzene. This second hypotheticaldegradation mechanism is also plausible, because humus contains manyaromatic molecules. It is conceivable that enzymes that degrade thearomatics in humus are not specific and are additionally capable ofconverting other aromatics, such as benzene.

Not excluded is the possibility that in the humic acid mixture,components are present that are necessary as vitamin for thebiosynthesis of enzymes of the anaerobic hydrocarbon-degrading bacteria.

It has been found that also in the absence of humic acids, allingredients remain in solution, without necessitating the use of achelating agent.

The invention can be very suitably used for cleaning soils andgroundwater contaminated with aromatic hydrocarbons. Examples of veryappropriate locations of use are locations where mineral oil has beenextracted or stored, the petrochemical industry, chemical industriallocations where benzene is used in production processes, and (former)gas stations.

Very suitably, the invention can be used for the degradation of benzene.This is surprising, since it is generally supposed that benzene is themost notorious of all aromatic soil contaminants, that is, mostdifficult to break down (see, for instance, Suarez and Rifai,Bioremediation Journal 3(4)(1999) 337-362).

In addition to benzene, according to the invention, other aromatics suchas BTEX (benzene, toluene, ethylbenzene and/or xylene), polycyclicaromatic hydrocarbons (PAHs), in particular naphthalene andphenanthrene, can be degraded very effectively. Also substitutedaromatics, in particular chlorinated aromatics, can be degradedaccording to the invention. Highly eligible for degradation according tothe invention are chlorinated benzenes, in particular monochlorobenzene.

The present invention can also be used for stimulating the anaerobicdegradation of aliphatic hydrocarbons, including alkanes and alkenes.Alkanes and alkenes are the most important components of oil and aretypically present as combination contamination with aromatichydrocarbons. Preferably, the soil-contaminating aromatic hydrocarbonsaccording to the invention comprise BTEX.

In principle, the invention can be used for degrading all aromaticcompounds, including the aromatics (that is, hydrocarbons having atleast one benzene ring) listed in the so-called blacklist published bythe Ministry of Health, Regional Development and the Environment(“Target Values and Intervention Values in Soil Remediation”, DutchGovernment Gazette, No. 39, 24 Feb. 2000, pp. 8-16), which list isunderstood to be incorporated herein.

The term “humic acids”, according to the conventional definition, refersto the water-soluble fraction of organic acids present in humus, or tothe salts (for instance the sodium salts) of these acids. The humicacids that are used according to the invention can be used in differentforms. Thus, it is possible to use purified humic acids, which can beobtained, for instance, through extraction of humus-rich products. Anadvantage of the use of (partly) purified humic acids is that, as aresult, a concentrated solution can be obtained, so that less liquidneeds to be injected. The humic acid can be used in the acid form or asa salt. Although a solution is normally easy to dose, it is alsopossible to make a powder mixture of the humic acid and the electronacceptor, and to introduce this into the soil in powder form, oroptionally as slurry. In this way, a very high concentration of humicacid and electron acceptor can be achieved.

In addition, it is possible to use the humic acid in the form ofcompost, humus-rich percolate and/or vegetable material. An advantage ofsuch humic acid-rich products is that they are cheaper.

As nitrate, for instance sodium, potassium or ammonium nitrate is used.Sodium and potassium nitrate enjoy preference because these are cheaper.Moreover, ammonium nitrate (fertilizer) is explosive and working with itis not always to be preferred in areas that are contaminated with thenormally easily flammable aromatic compounds.

The amount of humic acid and nitrate is preferably selected such thatthe concentration of humic acid in the location to be remediated is0.1-10 g/(liter of soil), more preferably 0.2-2 g/dm³, and theconcentration of nitrate (or other suitable electron acceptor) is 1-100mM, more preferably 5-50 mM (likewise based on the volume of soil).However, these concentrations may vary from one practical case toanother.

Working at a high concentration of humic acids and nitrate (or otherelectron acceptors) has as an additional advantage that a larger volumecan be treated per injection point. Even if the concentration directlyaround the injection point is so high as to be locally toxic to themicroorganisms, this still offers an advantage: through diffusion agradient will arise in the concentration of the injected substances,which gradient decreases in the direction away from the injection point.As a result, a larger “cloud” (that is, an area of a larger volume) canbe treated.

The relative weight ratio of humic acid/electron acceptor (based onsodium nitrate as electron acceptor) in a mixture according to theinvention is preferably about 2.

The invention further relates to a mixture comprising an aqueoussolution of humic acid and nitrate. Preferably, such a mixture contains1-10 wt. % of humic acid and 2-20 wt. % of nitrate (expressed as sodiumnitrate), more preferably 5-10 wt. % of humic acid and 10-20 wt. % ofnitrate, in particular 7-9 wt. % of humic acid and 12-18 wt. % ofnitrate. With a particular preference, the solution is as concentratedas possible. Such a mixture can be very suitable deployed in the methodaccording to the invention. If desired, this mixture can be supplementedwith additives. Suitable additives are vitamins, trace elements (Zn, Co,Cu, etc.) and/or macronutrients (S, P, Fe-sources) which improve thegrowth of the anaerobic bacteria. Normally, a standard vitamin mixtureand/or a standard trace mixture is used, as illustrated in the examplesbelow.

Preferably, the mixture according to the invention comprises one or moremacronutrients (each preferably in amounts of 0.05-10 g/dm³), one ormore trace elements (each preferably in amounts of 0.01-4 mg/dm³) and/orone or more vitamins (each preferably in amounts of 0.004-1 mg/dm³).

The macronutrients are preferably selected from (NH₄)₂SO₄, MgCl₂ 6H₂O,CaCl₂ 2H₂O, NaNO₃, KH₂PO₄, Na₂HPO₄, and combinations thereof.

The trace elements are preferably selected from EDTA, FeSO₄.7H₂O, ZnSO₄.7H₂O, MnCl₂ .4H₂O, H₃BO₃, CoCl₂ .6H₂O, CuCl₂ .2H₂O, NiCl₂ .6H₂O,Na₂MoO₄ .2H₂O, Na₂SeO₃ .5H₂O, Na₂WO₄ .2H₂O, and combinations thereof.

The vitamins are preferably selected from para-aminobenzoic acid,folinic acid, DT-lipoic acid, riboflavin, thiamin, nicotinic acid amide,pyridoxine.HCl, pantothenate, vitamin B₁₂, biotin and combinationsthereof.

According to the invention, the biological degradation of aromatics,including benzene, is stimulated and stabilized under anaerobicconditions. This provides advantages specifically in the treatment ofcontaminated locations at places that are difficult to treat withoxygen, such as the deep subsoil under buildings and in layers of clayand loam.

Because nitrate (or other electron acceptors) and humic acids are wellsoluble in water, in contrast to oxygen, it is possible to treatlocations with high concentrations of aromatics. The good solubility isan important advantage of the present invention.

An additional advantage is that humic acids promote the dissolution ofaromatics in water, in that humic acids have both hydrophobic andhydrophilic properties and so have a surfactant action. This promotesthe dissolution of undissolved aromatics (for instance present in thesoil in so-called floating layers, or in sediment layers), so that thesecan be degraded faster. Also aromatics that are sorbed into soilparticles (for instance clay particles) can dissolve more easily byvirtue of the presence of the humic acids. As a consequence, thecontaminant can be broken down and/or be pumped out of the soil at anaccelerated rate.

Instead of, or in addition to, the humic acids mentioned, also othercompounds with a quinone structure can be used, in particular compoundsthat contain an anthraquinone group, such as anthraquinone-2,6-disulfate(AQDS). Like humic acids, such compounds can be used as electron shuttleby anaerobic bacteria. However, since such compounds usually have a highcost price, humic acids are preferred according to the invention.

The invention will now be elucidated in and by an example andcomparative examples.

EXAMPLES

In a laboratory set-up, in a bioreactor at 20° C. and pH 7, an anaerobicmineral culture medium of the following composition was passed throughcontinuously (concentrations based on volume of the reactor, so-calledreservoir concentrations) Macronutrients (NH₄)₂SO₄ 0.5 g/l MgCl₂.6H₂O0.1 g/l CaCl₂.2H₂O 0.05 g/l NaNO₃ 1.7 g/l KH₂PO₄ 1.0 g/l Na₂HPO₄ 3.5 g/lTrace elements EDTA 1.0 mg/l FeSO₄.7H₂O 2.0 mg/l ZnSO₄.7H₂O 0.1 mg/lMnCl₂.4H₂O 0.03 mg/l H₃BO₃ 0.3 mg/l CoCl₂.6H₂O 0.2 mg/l CuCl₂.2H₂O 0.01mg/l NiCl₂.6H₂O 0.02 mg/l Na₂MoO₄.2H₂O 0.03 mg/l Na₂SeO₃.5H₂O 0.03 mg/lNa₂WO₄.2H₂O 0.03 mg/l Vitamins para-aminobenzoic acid 0.2 mg/l folinicacid 0.1 mg/l DT-lipoic acid 0.1 mg/l riboflavin 0.2 mg/l thiamin 0.4mg/l nicotinic acid amide 0.4 mg/l pyridoxine.HCL 1.0 mg/l pantothenate0.2 mg/l vitamin B₁₂ 0.2 mg/l biotin 0.004 mg/l

The dilution rate was 0.17 day¹. Benzene was continuously dosed to thereactor from a concentrated anoxic (that is: oxygen free) aqueoussolution with a spray pump, so that a concentration of 50-200 μM in thereactor (reservoir concentration) was obtained. In order to precludeoxygen formation by algae, the reactor vessel was darkened. In this way,a so-called chemostat culture was obtained. The reactor was inoculatedwith four nitrate-reducing and benzene-degrading enrichment culturesthat originated from different benzene-contaminated locations in theNetherlands.

Comparative Example 1

The above-mentioned solution was passed through the reactor togetherwith the benzene solution mentioned. No benzene degradation could bedetermined.

Comparative Example 2

The above-mentioned solution was supplemented with 5 mM acetate(reservoir concentration) and this solution was passed through thereactor together with the benzene solution in the same manner as inComparative Example 1. Again, no benzene degradation was determined.

Comparative Example 3

Comparative Example 2 was repeated, but now, instead of acetate,benzoate was added to the solution (reservoir concentration 5 mM).Again, no benzene degradation was determined.

Example 1 (Invention)

Comparative Example 3 was repeated, but now, after a period of 8 days, aswitch was made to a solution of 0.5 g/liter of sodium salt of humicacids (reservoir concentration, ex Sigma-Aldrich), which was dosed tothe reactor as described above. The benzene concentration (measured witha gas chromatograph) decreased rapidly: the half-life was ca. 1.5 days.The table below shows the course of the benzene concentration in time:Time/[days] Dosage Benzene concentration/[μM]²⁾ 0 Nitrate/benzoate 48.102.85 Nitrate/benzoate 51.49 7.05¹⁾ Nitrate/benzoate 51.38 13.86Nitrate/humic acids 29.41 15.03 Nitrate/humic acids 20.96 16.85Nitrate/humic acids 8.34 17.85 Nitrate/humic acids 2.47 20.84Nitrate/humic acids 1.73 28.85 Nitrate/humic acids 0.51 30.92Nitrate/humic acids 0.02 31.85 Nitrate/humic acids 0.01¹⁾moment after which a switch was made from a solution ofnitrate/benzoate to a solution of nitrate/humic acids.²⁾nominal concentration, that is, benzene in the total system based onthe liquid phase.

When subsequently nitrate was omitted from the medium, the nitrateconcentration decreased, until there was no nitrate to be measuredanymore. From that time, the benzene concentration in the reactor vesselincreased again. After addition of nitrate, the degradation processrecovered fast, resulting in complete benzene degradation within a week.

The process described in Example 1 (anaerobic benzene degradation in thepresence of humic acids and nitrate) was carried out and monitored for along time. After 18 months, still complete benzene degradation wasobserved under the above-mentioned conditions. Surprisingly, it wasestablished that dosing of oxygen (O₂) led to a strong increase of thebenzene concentration in the bioreactor, which indicates that thebenzene degradation stimulated by humic acids and nitrate is a strictlyanaerobic process.

These experiments demonstrate that the combination of humicacids/nitrate can be used for stimulating and stabilizing anaerobicdegradation of aromatics.

1. A method for the anaerobic biological degradation ofsoil-contaminating aromatic and/or aliphatic hydrocarbons present at acontaminated location, wherein a combination of one or more humic acids,if desired as salt, and at least one electron acceptor is added toanaerobic bacterial populations.
 2. A method according to claim 1,wherein said electron acceptor is selected from nitrogenous compounds,in particular nitrate, nitrite and/or N₂0; sulfate; chlorate;chlorinated hydrocarbons; and combinations thereof.
 3. A methodaccording to claim 2, wherein said electron acceptor is nitrate.
 4. Amethod according to claim 2, wherein said electron acceptor isperchloroethylene, trichloroethylene, 1,2-dichloroethane, chlorophenol,chlorobenzoic acid and/or chlorobenzene.
 5. A method according to claim1, wherein said location is a contaminated soil and wherein saidcombination of humic acids and electron acceptor is introduced into thesoil by means of injection.
 6. A method according to claim 1, whereinsaid aromatic hydrocarbons comprise BTEX (benzene, toluene, ethylbenzeneand/or xylene), polycyclic aromatic hydrocarbons (PAHs), aliphatichydrocarbons (alkanes, alkenes, oil), or mixtures thereof, whichhydrocarbons may or may not be halogenated.
 7. A method according toclaim 6, wherein said aromatic hydrocarbons comprise benzene which mayor may not be chlorinated, preferably monochlorobenzene.
 8. A methodaccording to claim 1, wherein said humic acids or salts thereof are usedin purified form and/or in the form of compost, humus-rich percolateand/or vegetable material.
 9. A mixture of humic acid and nitratecomprising an aqueous solution of 1-10 wt. % of humic acid and 2-20 wt.% of nitrate (expressed as sodium nitrate).
 10. Use of a mixtureaccording to claim 9, for the anaerobic biological degradation ofaromatic and aliphatic hydrocarbons.